A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

This method combines biochemical and behavioral approaches to help answer key questions in the neuroscience field about the impact of an acute kynurenine challenge in rats. The main advantage of the technique is that it provides a reliable and consistent method for the measurement of kynurenine pathway metabolites in rat brain and plasma. Demonstrating the procedure will be Carly Fabian, a student research assistant from my laboratory.

At the appropriate experimental end point after intraperitoneal kynurenine administration, thaw frozen harvested plasma on wet ice and dilute the thawed samples with ultrapure water. Transfer 100 microliters of diluted sample into individual containers of 25 microliters of 6%perchloric acid and vortex in centrifuge the samples. Then, transfer 100 microliters of supernatants from each tube into individual 250 microliter microcentrifuge tubes for kynurenine and kynurenic acid determination.

For kynurenic acid measurement from brain samples, thaw the frozen harvested brain samples on dry ice and individually weigh each sample on a precise analytical balance. When all of the samples have been weighed, move the tubes onto wet ice and dilute each tissue sample at a one to 10 weight by volume ratio with ultrapure water, followed by homogenization with a sonicator. Aliquot 100 microliters of each sample slurry to a new tube then add 25 microliters of 25%perchloric acid for vortexing and centrifugation.

Then, transfer 100 microliters of supernatant into individual 250 microliter microcentrifuge tubes for kynurenic acid determination. To prepare the high performance liquid chromatography, or HPLC, machine for kynurenine analysis, first place the individual HPLC intake tubes into the mobile phase, ultrapure water and 100%acetonitrile containers. Then, program the instrument to run 5%acetonitrile and 95%ultrapure water at 0.5 milliliters per minute and allow the HPLC machine to run for 20 to 30 minutes.

When a stable pressure and baseline have been achieved, change the solution composition to a 5%acetonitrile and 95%sodium acetate mobile phase and equilibrate the instrument for another 30 minutes. While the instrument is equilibrating, turn on the lamp in the fluorescence detector. Prior to the derivitization, pump the zinc acetate solution separately through a peristaltic pump that combines with the mobile phase post column and turn on the post column pump to a flow rate of 0.1 milliliters per minute for about 20 minutes, until it reaches a stable pressure of about 500 psi.

When the instrument is ready for the analysis, set the system software to control the sample sequence parameters and to allow the autoinjection of multiple samples. In the Run Sample'screen, click File'and drag down to Create New Sample Set'selecting Empty'in the new window. Individually list all of the standards and samples in the order they should be run, with the standards assayed at the beginning and end of the sequence.

In the function column, designate the standards and samples and set the program to inject water between every five samples. Set the run time for each sample to 15 minutes and inject 20 microliters of the samples from the standards, followed by 20 microliters of the plasma samples and 30 microliters of the brain homogenate samples as experimentally appropriate. Then, set the excitation and emission wavelengths according to the compound being measured and run the sequence.

To quantify the data, open the Browse Project'screen and under the Sample Sets'tab, double click on the sample set to be quantified. From the list of injections, highlight all of the standards and right click to select Process'in the new window. Then, use the drop-down menu to select Calibrate and Quantitate'Highlight all of the standards again, right click and select Review'When the chromatograms open, integrate and calibrate each standard to generate the standard curve.

Then, return to the Sample Sets'tab and double click on the sample set to quantitate and review as demonstrated, integrating and quantitating each sample to output the concentration of each sample based on the previously created standard curve. HPLC analysis of tissue samples harvested from kynurenine administered animals reveal a kynurenine retention time of about six minutes and a kynurenic acid retention time of about 11 minutes. The quantification of plasma kynurenine and hippocampal kynurenic acid reveals that acute kynurenine injection increases brain kynurenic acid levels with a peak achieved at two hours post-injection that returns to baseline at four hours post-injection.

Although no group differences are observed during passive avoidance paradigm training trials, control animals display a significant increase in latency to enter the dark side during testing trials, indicating contextual learning, while animals injected with kynurenine 24 hours previously do not display the same increase in latency, demonstrating a deficit in learning. Further, assessment of the impact of an acute kynurenine elevation during the light phase on sleep-wake architecture indicates a reduction in the total rapid eye movement sleep duration during the first two hours after kynurenine injection. These data are mirrored by an increase in wake duration during these time periods and a slight reduction in non-rapid eye movement sleep, demonstrating that an acute kynurenine elevation also causes disturbances in sleep-wake dynamics.

Following this procedure, other methods like behavioral analysis can be performed to answer additional questions about the role of elevated kynurenic acid formation in modulating cognition and sleep.